None.
No Federally sponsored research and development was used in making this invention.
Since they were first reported by Nuzzo and Allara in 1983, self-assembled monolayers (SAMs) composed of sulfur-terminated organic molecules adsorbed on and adherent to gold surfaces have shown broad utility in lubrication, electrochemistry, electronic and vibrational spectroscopy, photochemistry, diagnostics, the modification of biochemical membranes, catalysis, drug delivery, and facile modification of the absorptive properties of surfaces. (R. G. Nuzzo and D. L. Allara. Adsorption of bifunctional organic disulfides on gold surfaces. J. Am. Chem. Soc. 1983; 105: 4481-4483.) More recently, organic modifications of gold surfaces by SAMs have proven to be successful in nanotechnological biosensor applications, e.g., in commercially available chips for biomolecular interaction analysis with surface plasmon resonance. (S. L{haeck over (o)}fxc3xa5s, B. Johnsson, K. Tegendahl, and I. R{haeck over (o)}nnberg. Colloids Surf. B 1993; 1: 83-89.)
For example, Dijksma and coworkers have reported that an electrochemical immunosensor composed of self-assembled monolayers of cysteine or N-acetylcysteine on gold electrodes is useful for the detection of interferon-xcex3 at the attomolar level. (M. Dijksma, B. Kamp, J. C. Hoogvliet, and W. P. van Bennekom. Development of an electrochemical immunosensor for direct detection of interferon-xcex3 at the attomolar level. Analyt. Chem. 2001; 73: 901-907.) Similarly, Darder and coworkers have found that horseradish peroxidase retained its activity when immobilized onto a gold surface via a 3-thiopropionate tether and was useful as a peroxide biosensor. (M. Darder, K. Takeda, F. Pariente, E. Lorenzo, and H. D. Abruxc3x1a. Dithiobissuccinimidyl propionate as an anchor for assembling peroxidases at electrodes surfaces and its application in a H2O2 biosensor. Analyt. Chem. 1999; 71: 5530-5537.)
Likewise, poly- and oligo(ethylene glycols) (PEGs or OEGs, respectively; Structure 1, where R1 is MeO or HO and R2 is OH) have found widespread use in a variety of biotechnological and commercial applications, including the preparation of surfactants, ion-conducting materials, and conjugates of low and high molecular weight molecules. Investigators have found that these glycols provide good anchors for biological and non-biological receptor/reporter molecules or for ligands for biological and non-biological chelation or binding sites. Moreover, both PEGs and OEGs are known to reduce the nonspecific binding of proteins and other bioactive molecules to the surface to which they are conjugated. PEG and OEG derivatives are ideal for these applications because they are inexpensive, water soluble, stable, nonantigenic and non-immunogenic, and commercially available in a wide range of molecular weight distributions.
R1xe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)xxe2x80x94CH2CH2xe2x80x94R2xe2x80x83xe2x80x83Structure 1
In addition, conjugation with more highly branched and dendritic poly- and oligo(ethylene glycols) has been reported to be useful for improving the stability of protein drugs. [(a) D. C. Tully and J. M. J. Frechet. Dendrimers at surfaces and interfaces: chemistry and applications. Chem. Commun. 2001; 1229-1239. (b) I. Fuke, T. Hayashi, Y. Tabata, and Y. Ikada. Synthesis of poly(ethylene glycol) derivatives with different branchings and their use for protein modification. J. Controlled Release 1994; 30: 27-34. (c) J. M. Harris, F. M. Veronese, P. Caliceti, and O. Schiavon, U.S. Pat. No. 5,932,462.]
The broad utility of both classes of reagents (i.e., SAMs and PEGS or OEGS) suggests that synergistic benefits would obtain if libraries of reagents were available that combined the beneficial attributes of a SAM with those a PEG or OEG and exhibited additional features, such as the presence of reactive or activated groups at one end of each PEG or OEG chain. This combination of attributes would enable attachment of one terminus of such a combined SAM-forming-OEG reagent to a metal surface, yielding a SAM-OEG reagent, and attachment of a biological or non-biological receptor, ligand or reporter moiety at each of the other activated or reactive termini of the combined SAM/OEG reagent. The literature reports that describe examples of combined SAM/OEG reagents are limited to disclosures of methods of synthesis of OEG conjugates of linear alkyl monothiols and the effects of structure on the stability and physico-chemical properties of the reagents and the SAMs formed from them. (S. Svedhem, C-A. Hollander, J. Shi, P. Konradsson, B. Liedberg, and S. C. T. Svensson. Synthesis of a series of oligo(ethylene glycol)-terminated alkanethiol amides designed to address structure and stability of biosensing surfaces. J. Org. Chem. 2001; 66: 4494-4503.) Thus, the known reagents are limited to alkyl monothiols that lack an activated or reactive terminus at the end of the OEG chain and other desirable attributes that would enhance their utility.
Clearly, significant biotechnological advances in a spectrum of areas would be possible if activated or reactive, oligo(ethylene glycol)-terminated reagents and OEG-terminated reagents conjugated with a biological or non-biological receptor, ligand or reporter moiety useful for preparing self-assembled monolayers on gold were available. The present invention addresses this need.
The invention is based upon the recognition that the availability of activated or reactive, oligo(ethylene glycol)-terminated dithiolane compositions suitable for use in preparing self-assembled monolayers on a metal would enable significant advances in the biotechnological arts.
Thus, the invention provides highly versatile tethers suitable for immobilization on a metal backbone, wherein one segment of the tether is a linear or branched oligo(ethylene glycol) residue and the other segment of the tether is an alkyl-substituted 1,2-dithiolane. Further, one terminus of each oligo(ethylene glycol) residue is activated or reactive, enabling the preparation of conjugates of the oligo(ethylene glycol)-terminated dithiolane compositions that are also suitable for immobilization on a metal backbone.
One embodiment of the present invention comprises linear or branched oligo(ethylene glycol)-terminated 3-alkyl-1,2-dithiolanes having the formula: 
wherein m is from about 3 to about 20; n is from 2 to about 6; OEG is shorthand for a linear oligoether having the general structure xe2x80x94(CH2CH2O)xxe2x80x94 wherein x is from 2 to about 100, or for a branched oligoether wherein each branch comprises a linear oligoether having this general structure; one terminus of the OEG residue is covalently joined to the terminus of the alkyl side chain of the dithiolane by a linker L, wherein L is N, O, S, P, or an amide or hydrazide group; and each of the other termini of the OEG residue is a reactive or activated substituent Z that can be joined covalently to a biological or non-biological, ligand, sequestering, or reporter moiety. Examples of suitable reactive or activated substituents Z include an amino, guanidino, sulfhydryl, or activated ester moiety; a substituent that is reactive toward nucleophilic displacement, such as chloride, bromide, iodide, tosylate, tresylate, or mesylate; a group that is reactive toward nucleophilic addition, such as cyanate, isocyanate, thiocyanate, isothiocyanate, maleimide, oxirane, thiirane, or azirane; a carbonyl group; or a hydroxyl group.
A preferred embodiment comprises oligo(ethylene glycol)-terminated thioctic acid derivatives having the formula: 
wherein n is from 2 to about 6; the symbol OEG is a linear oligoether having the general structure xe2x80x94(OCH2CH2)xxe2x80x94 and x is from 2 to about 100, or is a branched oligoether wherein each branch comprises a linear oligoether having this general structure; one terminus of the OEG residue is covalently joined to the alkyl side chain of thioctic acid by a linker L, wherein L is amide or hydrazide; and each of the other termini of the OEG residue is a reactive or activated substituent Z that can be joined covalently to a biological or non-biological ligand or reporter moiety.
A particularly preferred embodiment comprises oligo(ethylene glycol)-terminated d-thioctic acid derivatives having the formula: 
wherein n is from 2 to about 6; the symbol OEG is a linear oligoether having the structure xe2x80x94(OCH2CH2)xxe2x80x94 and x is from 2 to about 100, or is a branched oligoether wherein each branch comprises a linear oligoether having this structure; one terminus of the OEG residue is covalently joined to the alkyl side chain of d-thioctic acid by a linker L, wherein L is amide or hydrazide; and each of the other termini of the OEG residue is a reactive or activated substituent Z that can be joined covalently to a biological or non-biological ligand or reporter moiety.
Another embodiment of the present invention comprises oligo(ethylene glycol)-terminated 4-alkyl-1,2-dithiolanes having the formula: 
wherein m is from 3 to about 20; n is from 2 to about 6; the symbol OEG is a linear oligoether having the structure xe2x80x94(OCH2CH2)xxe2x80x94 and x is from 2 to about 100, or is a branched oligoether wherein each branch comprises a linear oligoether having this structure; one terminus of the OEG residue is covalently joined to the terminus of the alkyl side chain of the dithiolane by a linker L, wherein L is N, O, S, P, or an amide, or hydrazide; and each of the other termini of the OEG residue is a reactive or activated substituent Z that can be joined covalently to a biological or non-biological ligand or reporter moiety. Examples of suitable reactive or activated substituents Z include an amino, guanidino, sulfhydryl, or activated ester moiety; a substituent that is reactive toward nucleophilic displacement, such as chloride, bromide, iodide, tosylate, tresylate, or mesylate; a group that is reactive toward nucleophilic addition, such as cyanate, isocyanate, thiocyanate, isothiocyante, maleimide, oxirane, thiirane, or azirane; a carbonyl group; or a hydroxyl group.
Also provided in accordance with the invention are conjugates of these activated polymers with a biological or non-biological receptor, ligand, sequestering, or reporter moiety such as a polypeptide, protein, enzyme, phospholipid, lipid, liposome, nucleoside, oligonucleotide, drug, dye, antibody reporter molecule, ligand, cyclodextrin, carceplex, boronate, biological membrane, or a surface of a solid material that is compatible with living organisms, tissue, or fluids. Further provided are methods for preparation of these conjugates.
Also provided in accordance with the invention is a self-assembled monolayer (SAM) composition comprising an activated or reactive, OEG-modified-1,2-dithiolane composition or a conjugate of an OEG-modified-1,2-dithiolane composition adherent to gold, silver, copper, mercury, or an amalgam of these metals. A SAM composition comprising an activated or reactive, OEG-modified-1,2-dithiolane composition or a conjugate of an OEG-modified-1,2-dithiolane composition adherent to gold is most preferred. Further provided are methods for the preparation of these self-assembled monolayers and methods for their dissociation.
The unexpected utility of an activated or reactive, oligo(ethylene glycol)-terminated 1,2-dithiolane composition of the present invention or a conjugate of a reactive, OEG-terminated 1,2-dithiolane composition of the present invention as compared to the utility of the linear OEG-terminated, linear alkyl monothiols known in the art is believed to come from five sources. First, the 1,2-dithiolane segment of a 1,2-dithiolane composition of the present invention reacts with gold or another metal of the present invention to provide a self-assembled monolayer (SAM) composition that is stabilized by multiple sulfur-metal bonds. The multiple sulfur-metal bonds render the resulting SAM composition more stable than that of a monothiol. Second, the other segment of a 1,2-dithiolane composition of the present invention presents at least one activated or reactive terminus available for binding a biological or non-biological receptor, ligand, sequestering, or reporter moiety, or presents at least one terminus to which a biological or non-biological receptor, ligand, sequestering, or reporter moiety may be bound covalently. Third, when bound to the metal surface, a 1,2-dithiolane composition of the present invention is chemically stable in a wide variety of hostile media and conditions. This stability enables presentation of at least one biological or non-biological receptor, ligand or reporter moiety and capture and/or extraction and/or sequestering of a species of interest from a complex environment without undesirable dissociation of the oligo(ethylene glycol)-terminated dithiolane-metal complex during exposure to the hostile environment. Fourth, each of the opposing termini at the end of the OEG-portion of a 1,2-dithiolane composition of the present invention is reactive with, or may be activated to be reactive with, any one of a broad spectrum of electrophilic or nucleophilic reagents. This reactivity enables covalent attachment of a biological or non-biological receptor, ligand, sequestering, or reporter moiety to an activated or reactive, oligo(ethylene glycol)-terminated 1,2-dithiolane composition of the present invention either prior to its attachment to a metal or following its attachment to a metal. Further, if the OEG-portion of a 1,2-dithiolane composition of the present invention is branched, each activated or reactive terminus of an OEG-branch may be joined covalently to a biological or non-biological receptor, ligand or reporter moiety, thereby enabling presentation of a plurality of ligand or reporter moieties. Presentation of a plurality of a biological or non-biological receptor, ligand or reporter moieties is believed to enable more effective binding of a species of interest and its sequestration from a complex environment. Fifth, each composition of the present invention presents a moderately hydrophilic surface (i.e., the OEG-portion of a composition of the present invention) to the external environment. Monolayers of poly- or oligo(ethylene glycol) derivatives are known to minimize non-specific binding of biomolecules to the interactive terminus of the SAM. (C. Pale-Grosdemange, E. S. Simon, K. L. Prime, and G. M. Whitesides. Formation of self-assembled monolayers by chemisorption of derivatives of oligo(ethylene glycol) of structure HS(CH2)11(OCH2CH2)mOH on gold. J. Am. Chem. Soc. 1991; 113: 12-20.)
In addition to the five utilities cited above, a sixth utility has not been heretofore recognized by skilled artisans and applies particularly to the 1,2-dithiolane compositions of the present invention. Application of electrical voltage to a gold-sulfur-terminated reagent complex is known to effect the severance of the gold-sulfur reagent bond and release the reagent as a thiol. With respect to an OEG-terminated 1,2-dithiolane composition of the present invention, application of voltage to a gold-complex of a 1,2-dithiolane composition of the present invention severs both gold-sulfur bonds and releases the composition as the dithiol. Surprisingly, the inventor has found that this dithiol rapidly oxidizes to a ring-closed disulfide (i.e., a 1,2-dithiolane of the present invention).
This unexpected and rapid ring closure to a 1,2-dithiolane composition of the present invention offers distinct advantages to users of the present invention. One significant advantage relates to the relative nucleophilicity and reactivity of thiols compared to the nucleophilicity and reactivity of disulfides. Thiols are nucleophiles, and can undergo a variety of reactions, including, for example, the displacement of another thiol that is part of a disulfide. Thus, release of a thiol enables undesirable displacement reactions to occur, reactions that destroy (i.e., xe2x80x9cscramblexe2x80x9d) existing disulfide bonds that may be critical to the structure and activity of a protein and cause its inactivation or denaturation. (Insulin is an example of a protein in which maintenance of the native disulfide bonds is critical. If insulin is exposed to a thiol, xe2x80x9cscramblingxe2x80x9d of the internal disulfide bonds takes place, and the protein is inactivated.) In contrast, after release from a SAM composition of the present invention, a 1,2-dithiolane of the present invention is re-formed. The disulfide (i.e., 1,2-dithiolane) thus formed is not a nucleophile and does not cause displacement reactions. The lack of chemical reactivity of the 1,2-dithiolane segment of a 1,2-dithiolane of the present invention is advantageous to the user of the present invention in a number of ways, including, by way of example, enabling monitoring of a 1,2-dithiolane composition of the present invention by surface plasmon resonance or mass spectrometry.
A seventh advantage of the 1,2-dithiolanes of the present invention relates specifically to the embodiments in which the 1,2-dithiolane is thioctic acid, d-thioctic acid or a derivative thereof. d-Thioctic acid is a natural substance found in mammals and is an important biological anti-oxidant and enzyme co-factor. Since some of the 1,2-dithiolanes of the present invention are derivatives of d-thioctic acid, it is reasonable to anticipate that these dithiolanes will be physiologically compatible. This is advantageous to the user of the present invention in a number of ways, including, by way of example, enabling use of such a 1,2-dithiolane of the present invention as a means for drug delivery.